BackgroundMitotic spindle apparatus (MSA) antibodies are rare findings with undefined clinical significance in clinical research. We aimed at investigating the prevalence and clinical significance of anti‐MSA antibodies in Chinese population.MethodsBetween 2008 and 2013, a total of 180,180 patients were studied for the presence of anti‐MSA antibodies. The clinical details and laboratory data of anti‐MSA‐positive patients were retrospectively collected and analyzed.ResultsOf the 180,180 patients tested, 68,640 patients presented with positive antinuclear antibodies (ANAs, 38.10%), but only 32 patients with positive anti‐MSA antibodies (0.018%). Diagnoses were established in 22 of 32 patients: 16 connective tissue diseases (CTDs), mainly Sjogren syndrome (SS, 5/16), rheumatoid arthritis (RA, 4/16), and systemic lupus erythematosus (SLE, 3/16), and 6 nonautoimmune conditions. The most frequent clinical symptoms of the anti‐MSA‐positive patients were arthralgia and eyes and mouth drying. Additionally, 70% of anti‐MSA antibodies were not associated with other ANAs, however, when associated, the most frequent ANA was anti‐SSA.ConclusionsAnti‐MSA antibodies have a low prevalence and female gender predominance. Anti‐MSA antibodies are primarily associated with CTDs, mainly SS, RA, and SLE. The presence of anti‐MSA antibodies might be the unique serological markers of the CTDs, especially when anti‐SSA, SSB, and dsDNA antibodies are negative, or the level of RF is low.